Project Summary The first critical step of HIV-1 infection is fusion of viral and target cell membranes mediated by envelope glycoprotein (Env). The mature Env spikes [trimeric (gp160)3, cleaved to (gp120/gp41)3] are the sole antigens on the virion surface. Conformational changes in gp120 when triggered by binding to receptor (CD4) and co- receptor (e.g., CCR5 or CXCR4) lead to a cascade of refolding events in gp41, and ultimately to membrane fusion. Vast amount of structural information is available for the ectodomain of Env, but much less is known regarding its transmembrane domain (TMD) and its membrane-proximal (MP) regions, including the membrane proximal external region (MPER) and the cytoplasmic tail (CT). We recently made an unexpected discovery that truncation of the CT domain drastically reshapes the antigenic surfaces of the Env ectodomain on the other side of the membrane. Deep understanding of the physical coupling (conformation and/or dynamics) between the CT and the ectodomain mediated by the TMD may guide Env-based immunogen design to induce broadly neutralizing antibodies (bnNabs). We thus hypothesize that the membrane-interacting domains (MPER, TMD and CT) of HIV-1 Env adopt defined structures which are critical for its stability, function and antigenicity. We have already completed a TMD structure at the atomic resolution using the state-of-the- art NMR technology. When reconstituted in bicelles that mimic lipid bilayer, the TMD forms a well-ordered trimer mainly stabilized by a tightly packed hydrophilic core near its C-terminal end that can potentially be influenced by the CT domain. In this application, we plan to combine structural biology approaches and functional assays to elucidate the roles of the membrane-interacting domains of HIV-1 Env. We will purse the following specific aims: 1) we will investigate TMD assembly of HIV and SIV Env and possible interaction with the fusion peptide; 2) we will provide structural information for the MP regions of Env in the context of membrane; 3) we will elucidate the role of the membrane-interacting domains of Env in its stability, function and antigenicity; 4) we will design trimer immunogens to mimic the native and functional HIV-1 Env spike..